Endothelin-1 (ET-1) can cause osteoblastic bone metastases, stimulating dysregulated new bone formation by activating the endothelin A receptor (ETAR) on the osteoblast. The role of ET-1 in normal bone development, growth and remodeling is unclear. Our preliminary data indicate that: 1) ET-I is a potent stimulator of osteoblast activity and new bone formation in vivo; 2) Tumor-produced ET-1 stimulates osteoblastic metastases via ETAR; 3) ETAR blockade has sex steroid-dependent effects on bone mass: most strikingly, ETAR blockade reduces bone mass in hypogonadal but increases bone mass in intact female mice. 4) ET-1 increases expression of bone active factors IL-6 and Cyr61 and decreases that of Dkkl in osteoblasts, while increasing expression of the transcription factors C/EBPdelta and TSC-22. ET-1 did not alter EGFR activation or VEGF. The following hypotheses will be tested: 1) ET-1 plays an important role in normal bone remodeling. 2) ET-1 mediates effects on osteoblasts by changing the secretion of paracrine regulators 1L-6, Dkk1 and potentially Cyr61. 3) ET-1 increases the osteoblast transcription factor C/EBP\delta that interacts with Runx2, and TGFbeta-regulated TSC-22. 4) ETAR blockade results in reduced osteoblastic bone formation and increased osteoclast activity and may accelerates the osteoclastic bone resorption and subsequent bone loss associated with sex steroid deficiency. 5) Estrogen suppresses ET-1 synthesis. In estrogen-deficient states, increased ET-1 maintains bone mass Three Specific Aims are proposed: Aim 1: Determine the role of ET-1 and ETAR in normal bone development, growth, and remodeling 1A. Determine bone phenotype of mice with ETAR-null osteoblasts 1B. Determine bone phenotype of mice with ET-l- overexpressing osteoblasts Aim 2: Determine the role of ET-1 and ETAR in bone remodeling in sex-steroid deficient states 2A. Determine interactions between sex steroids and ET-1 in osteoblastic new bone formation 2B. Determine effects of combined inhibition of resorption and ETAR signaling on bone mass 2C. Determine bone phenotype of hypogonadal mice with ETAR-null or ET-1 ++g osteoblasts Aim 3: Determine the molecular mechanisms of ET-1 effects on bone 3A. Determine if ET-1 mediates its effects on osteoblasts via secreted IL-6, Dkk1, or Cyr61 3B. Determine effects of ET-1 on osteoblast transcription factors C/EBPdelta and TSC-22 3C. Determine major signaling pathways activated by ET-1 in osteoblasts 3D. Assess the interactions of sex steroids with ET-1 in bone cells The physiological role of ET-1 and ETAR in normal bone homeostasis needs investigation. 1, ETAR blockade is in clinical trials for many disease states, but the consequences for bone are unknown. Cancer patients treated with chronic ETAR blockade may suffer increased bone loss, in addition to that caused by sex-steroid deficiency. We will define the mechanisms and determine if this effect can be attenuated by bisphosphonate treatment. 2, an understanding of ET-1 activation of ETAR in the osteoblast may lead to new anabolic therapies for low bone mass. The experiments proposed will use in vivo and in vitro approaches to test the role of ET-1 activation of osteoblast ETAR in bone growth and remodeling.